质体RNA聚合酶的结构研究

Plastids are semi-autonomic organelles in plant cells and retain a genome that originates from the symbiotic photosynthetic bacteria. Their genome transcription, which is vital for plant survival, is carried out by two types of RNA polymerase. The single-unit nucleus-encoded polymerase (NEP) is a phage-type RNA polymerase and initiates plastid genome transcription; the multiple-unit plastid-encoded polymerase (PEP) is a bacterial type RNA polymerase and essential for plastid development and function. NEP and PEP coordinate the genome transcription, but their recognition mechanisms and preferences of promoters remain unclear. Elucidation of their molecular basis awaits three-dimensional structural determination. The sequences of NEP and PEP have notable differences from their homologs with known structure, particularly for the plant/cyanobacterium-specific PEP subunits, whose long insertion sequences lack any structural information. With the rapid advance of molecular plant biology, our proposed structural study of plastid RNA polymerases will contribute to the exploration of plastid and the development of plastid biotechnology. An understanding of the molecular basis for plastid genome transcription can also help depicting the regulatory networks within this organelle and provide theoretical support to the use of plastid transformation technology.

质体源自内共生的光合细菌，是植物细胞中具有半自主性的细胞器，具有独立的基因组。质体基因组的转录对于植物的生存具有极其重要的作用，由两种RNA聚合酶执行：细胞核编码的单亚基的NEP和质体基因编码的多亚基的PEP。NEP被称为噬菌体型RNA聚合酶，是质体基因转录的起始；PEP被称为细菌型RNA聚合酶，在质体发育和承担功能的过程中起着关键作用。两种RNA聚合酶共同作用转录质体基因组，但它们如何识别启动子及转录偏好性等问题仍有赖三维结构信息的获得。NEP和PEP的序列分别与已有同源结构的序列有较大差异，特别是PEP具有光合细菌和植物特有的亚基组成，其独特的长插入序列入尚无任何结构信息。随着质体研究领域和分子植物技术的迅速发展，质体RNA聚合酶的结构解析将对这一领域和相关技术产生极大的促进。在分子水平阐明质体内的转录机制将有助于揭示质体基因表达的调控网络，并将为基于质体转化的生物技术提供理论支撑。

质体起源于内共生的原核光合细菌，质体编码的RNA聚合酶（RNAP）与蓝细菌的RNAP具有很高的序列同源性，因而对后者结构的解析可以为前者的研究提供重要信息。本项目采用单颗粒冷冻电镜技术，分别解析了嗜热蓝细菌RNAP（TvRNAP）核心酶和结合DNA双链的全酶结构。TvRNAP核心酶包括alpha、beta、beta'、gamma、omega亚基，其结构揭示了蓝细菌与质体RNAP特有的、位于beta'亚基的大段插入序列如何折叠，提示该序列在转录过程中发挥调节功能。DNA双链分开形成的转录泡与TvRNAP全酶（即核心酶与sigma因子的复合物）之间的相互作用在结构中被清晰展现。TvRNAP的结构已存入蛋白质数据库（PDB）。此外，在本项目的资助下，项目组培养博士3人，硕士3人，发表论文6篇，发布PDB坐标11套。